DNA purification and isolation using magnetic particles

• US 5,705,628 A
• Filed: 09/20/1994
• Issued: 01/06/1998
• Est. Priority Date: 09/20/1994
• Status: Expired due to Term

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Hawkins, Trevor
• Primary Examiner(s): Kunz, Gary L.
• Application Number:
  US08/309,267
• Time in Patent Office: 1,204 Days
• Field of Search: 536/25.4, 435/91.1, 435/975, 436/526, 252/62.51
• US Class Current: 536/25.4
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: Y10S 435/975 Kit

Abstract

A method of separating polynucleotides, such as DNA, RNA and PNA, from a solution containing polynucleotides by reversibly and non-specifically binding the polynucleotides to a solid surface, such as a magnetic microparticle, having a functional group-coated surface is disclosed. The salt and polyalkylene glycol concentration of the solution is adjusted to levels which result in polynucleotide binding to the magnetic microparticles. The magnetic microparticles with bound polynucleotides are separated from the solution and the polynucleotides are eluted from the magnetic microparticles.

Claims

1. A method of reversibly binding DNA non-specifically to magnetic microparticles having a carboxyl group-coated surface, comprising the steps of:

a) combining magnetic microparticles having a carboxyl group-coated surface to a solution containing DNA; and

b) adjusting the salt and polyethylene glycol concentration of the solution to a concentration suitable for binding the DNA onto the surfaces of the magnetic microparticles,

thereby binding the DNA non-specifically to the magnetic microparticles.

2. The method of claim 1, wherein the polyethylene glycol has a molecular weight between about 6000 and about 10,000, and wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride.

3. The method of claim 2, wherein the polyethylene glycol has a molecular weight of about 8000 and the salt is sodium chloride.

4. The method of claim 3, wherein the concentration of the polyethylene glycol is adjusted to between about 5% and about 15% and wherein the concentration of sodium chloride is adjusted to between about 0.5M and about 5.0M.

5. The method of claim 4, wherein the concentration of the polyethylene glycol is about 10% and the concentration of sodium chloride is about 1.25M.

6. The method of claim 4, wherein the DNA solution is a cleared lysate.

7. The method of claim 4, wherein the DNA solution comprises the reaction product of a PCR amplification.

8. The method of claim 4, wherein the DNA solution comprises M13 phage DNA, wherein the host cells have been removed.

24. A method of determining the nucleotide sequence of a DNA molecule, comprising binding the DNA molecule non-specifically to magnetic microparticles having a carboxyl group-coated surface by the method of claim 4 and sequencing the DNA while the DNA is bound to the magnetic microparticles.

9. A method of separating DNA from a solution containing DNA, comprising the steps of:

a) combining magnetic microparticles having carboxyl group-coated surfaces and a solution containing DNA, thereby producing a first combination;

b) adjusting the salt concentration and the polyethylene glycol concentration of the first combination to concentrations suitable for binding DNA to surfaces of the magnetic microparticles, thereby producing a second combination comprising DNA bound non-specifically to magnetic microparticles;

c) separating the magnetic microparticles from the second combination;

d) contacting the magnetic microparticles separated in c) with the bound DNA in an elution buffer, whereby DNA is dissolved in the elution buffer and DNA bound from the magnetic microparticles is separated from the magnetic microparticles; and

e) separating the magnetic microparticles from the elution buffer.

10. The method of claim 9, wherein the separation of the magnetic microparticles in steps b) and d) is done magnetically.

11. The method of claim 10, wherein the DNA bound to the magnetic microparticles is washed with a buffer solution, wherein the buffer solution dissolves impurities bound to the magnetic microparticles while leaving the DNA bound to the magnetic microparticles.

12. The method of claim 10, wherein the DNA bound to the magnetic microparticles is washed with a selective elution solution, wherein the selective elution solution dissolves small DNA fragments bound to the microparticles while leaving large DNA fragments bound to the magnetic microparticles.

13. The method of claim 12, wherein the DNA dissolved by the elution solution is isolated.

14. The method of claim 11, wherein the DNA is digested by one or more enzymes while bound to the magnetic microparticles.

15. The method of claim 11, wherein the DNA solution of step a) is a cleared lysate.

16. The method of claim 11, wherein the DNA of the DNA solution of step a) is the reaction product of a PCR amplification procedure.

17. The method of claim 11, wherein the DNA solution of step a) comprises M13 phage DNA, wherein the host cells have been removed.

18. The method of claim 11, wherein the DNA solution of step a) comprises DNA of interest, wherein the DNA of interest has been separated from undesired DNA by electrophoresis in an agarose gel, and a melted agarose plug excised from the electrophoresis gel, wherein the agarose plug contains the DNA of interest.

19. A method of separating DNA from a solution containing DNA, comprising the steps of:

a) combining a solution containing DNA and carboxyl group-coated magnetic micro-particles, thereby producing a first combination;

b) combining the first combination with sufficient quantities of polyethylene glycol and sodium chloride to produce a second combination having a final polyethylene glycol concentration of from about 5% to about 15% and a final sodium chloride concentration of from about 0.5M to about 5.0M, whereby DNA in the solution binds nonspecifically to the magnetic microparticles, producing magnetic microparticles having DNA bound thereto;

c) separating magnetic microparticles from the second combination, thereby producing separated magnetic microparticles having DNA bound thereto;

d) contacting the separated magnetic microparticles having DNA bound thereto with an elution buffer, whereby the DNA is released from the magnetic microparticles and an elution buffer containing DNA and magnetic microparticles is produced; and

e) separating DNA released from the magnetic microparticles from the elution buffer.

20. The method of claim 19, wherein the polyethylene glycol has a molecular weight of between about 6000 and about 10,000 and wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride.

21. The method of claim 20, wherein the polyethylene glycol has a molecular weight of about 8000 and the salt is sodium chloride.

22. The method of claim 21, wherein the concentration of the polyethylene glycol is adjusted to between about 5% and about 15% and wherein the concentration of sodium chloride is adjusted to between about 0.5M and about 5.0M.

23. The method of claim 22, wherein the concentration of the polyethylene glycol is about 10% and the concentration of sodium chloride is about 1.25M.

25. A kit comprising a binding buffer and magnetic microparticles with carboxyl group-coated surfaces, wherein the binding buffer comprises a suitable salt and a suitable polyethylene glycol at a concentration suitable for binding DNA to the surface of the magnetic microparticles.

26. The kit of claim 25, wherein the kit further comprises a suitable elution buffer, wherein the elution buffer is capable of dissolving DNA bound to the magnetic microparticles.

27. The kit of claim 26, wherein the kit additionally comprises a wash buffer, wherein the wash buffer is capable of dissolving impurities bound to magnetic microparticles but is incapable of dissolving DNA bound to magnetic microparticles.

28. The kit of claim 26, wherein the kit additionally comprises reagents necessary for clearing a cell lysate.

29. A method of isolating polynucleotides from a solution containing a mixture of biomolecules, comprising the steps of:

a) combining a solution containing a mixture of biomolecules with carboxyl-coated magnetic microparticles;

b) adjusting the polarity and the hydrophobicity of the resulting solution so as to adsorb polynucleotides onto the surface of the microparticles;

c) separating the microparticles from the solution containing the biomolecules;

d) combining the separated magnetic microparticles with a second solution whose polarity and hydrophobicity results in the polynucleotides being desorbed from the surface of the magnetic microparticle, and dissolved in the second solution; and

e) separating the magnetic microparticles from the second solution.